The present invention relates to proteins having immunomodulatory activity and therapeutic agents for immune diseases. More specifically, the present invention relates to proteins derived from protein produced from helminth, which modulate the immune system in a host.
Agents including steroids and immunosupressants, such as cyclosporine, have been used conventionally for treatment of autoimmune diseases, called intractable diseases, and allergic diseases. However, these agents have only symptomatic therapy and no effective agent exerting a markedly advantageous effect has been developed yet. Further, the steroids and cyclosporine have problems including strong side effects and drug resistance.
Recently, the dramatic advance of molecular biology and immunology has determined the detailed mechanism of immune system, various factors involved in this mechanism and receptors recognizing these factors, and has unfolded their functions and roles in the immune system. Examples of these factors include cytokines, such as various interleukins, receptors recognizing the cytokines, antibodies against the cytokines and receptors, adhesion molecules and antibodies against the adhesion molecules.
For treatment of autoimmune diseases and allergy, for example, many attempts have been made to use the above-mentioned factors as so-called xe2x80x9cbiopharmaceuticalsxe2x80x9d in the treatment where these diseases result from dysfunction of these factors.
However, these attempts are only directed to the treatment of factors functioning abnormally. These attempts therefore provide only a local immunological treatment and are considered to be an extension of conventional symptomatic treatments.
Accordingly, there is a need for a therapeutic agent based on new idea supported by the mechanism of immune system as a whole in order to cure the above intractable diseases completely.
On the other hand, parasitologists have found a tendency for patients infected with parasitic helminth to be less susceptible to allergy, based on the epidemiological correlation between helminth infection and allergy. They have also reported that patients with systemic lupus erythematosus exhibit ameliorated symptoms by parasitic infection.
However, some allergologists state that such an epidemiological correlation is groundless and absolutely unfounded because the tendency found by parasitologists has not been scientifically proved.
Regarding molecules derived from helminth, Fujita et al. has reported finding an allergen from Dirofilaria immitis (Fujita et al., 1979), and Horii et al. has found Dirofilaria immitis neutrophil chemotactic factor (DiNCF) and has determined its amino acid sequence (Horii et al., 1986). Further, C. B. Poole et al. has isolated DiNCF as Cuticular antigen produced from Dirofilaria immitis and has cloned its partial gene sequence, indicating that DiNCF has a structure in which the antigen molecules repeated in tandem (C. B. Poole, 1992).
The cDNA cloning of DiNCF has also been reported, for example, by J. Culpepper (J. Clupepper, 1992), Ohashi et al. (Ohashi et al., 1993), and C. B. Poole et al. (C. B. Peele et al., 1996).
These reports suggest nothing about the immunomodulatory activity of the helminth-produced molecules as defined in the present invention, because of focusing their attention only on neutrophil chemotactic activity and/or antigenicity of the molecules.
An extract of parasitic helminth has been already known to induce B cell proliferation. For example, a soluble molecule from Ascaris suum involved in IgE production (T. D. G. Lee et al., 1993), a crude antigen from Toxocara canis having the ability to increase human peripheral blood cells (Inuo et al., 1995) and an antigen from Ascaris suum having mitogenicity (T. D. G. Lee et al., 1995) have been reported.
However, each of these extracts was used without further isolation in each experiment, so that a single molecule isolated from the extracts has not been reported to induce B cell proliferation independently of T cells.
In view of the foregoing, there is still a lot of discussion among scientists of different fields and they have not reached a clear conclusion. We have focused our research effort on how the parasitic helminth infection affects the immune system in a host, and have proved that a molecule derived from parasitic helminth is effective in the treatment of immune diseases, and finally have completed the invention.
The present invention is based on the self-defense mechanism that parasites have attained over several hundreds of millions of years in order to protect themselves against immune response of their hosts. The present invention provides an agent designed according to this concept, i.e., a brand-new idea.
The present invention provides a protein of the following formula (I) having immunomodulatory activity:
X-Y-Zxe2x80x83xe2x80x83(I)
wherein X represents an amino acid sequence of SEQ ID NO: 1 or 2, each of Y and Z is absent or represents an amino acid sequence of SEQ ID NO: 1 or 2.
The present invention also provides the following recombinant protein (a) or (b):
(a) a protein having an amino acid sequence selected from SEQ ID NO: 7-14; and
(b) a protein having an amino acid sequence selected from SEQ ID NO: 7-14 in which one or more amino acids are deleted, substituted or added, and having immunomodulatory activity.
The present invention also provides the following recombinant protein (a) or (b):
(a) a protein having an amino acid sequence of SEQ ID NO: 15; and
(b) a protein having an amino acid sequence of SEQ ID NO: 15 in which one or more amino acids are deleted, substituted or added, and having immunomodulatory activity.
The present invention further provides an immunomodulating agent comprising the following recombinant protein (a) or (b):
(a) a protein having an amino acid sequence selected from SEQ ID NO: 1-6; and
(b) a protein having an amino acid sequence selected from SEQ ID NO: 1-6 in which one or more amino acids are deleted, substituted or added, and having immunomodulatory activity.
The present invention further provides a therapeutic agent for immune diseases, which comprises one or more proteins described above as an active ingredient.
The immune disease includes autoimmune diseases, in particular, Th1-dominant autoimmune diseases selected from the group consisting of multiple sclerosis, insulin-dependent diabetes mellitus, Crohn""s disease, uveitis, chronic rheumatism, and systemic lupus erythematosus.
The immune disease also includes autoimmune diseases not known to be Th1-dominant, which are selected from the group consisting of scleroderma, multiple myositis, vasculitis syndrome, mixed connective tissue disease, Sjxc3x6gren""s syndrome, hyperthyroidism, Hashimoto""s disease, myasthenia gravis, Guillain-Barrxc3xa9 syndrome, autoimmune hepatopathy, ulcerative colitis, autoimmune nephropathy, autoimmune hematopathy, idiopathic interstitial pneumonia, hypersensitivity pneumonitis, autoimmune dermatosis, autoimmune cardiopathy, autoimmune infertility, and Behcet""s disease.
The present invention also provides an agent for stimulating IgE production, which comprises one or more proteins described above as an active ingredient. The present invention further provides a therapeutic agent for allergic diseases, which comprises one or more proteins described above as an active ingredient.
The allergic disease includes chronic bronchitis, atopic dermatitis, pollinosis (allergic rhinitis), allergic angiitis, allergic conjunctivitis, allergic gastroenteritis, allergic hepatopathy, allergic cystitis, and allergic purpura.
The present invention also provides an immunomodulating agent which comprises one or more proteins described above as an active ingredient. The immunomodulating agent may modulate rejection reaction occurring in organ transplantation.
The present invention also provides an immunomodulation method which comprises administering one or more proteins described above in an effective amount to a patient in need of such treatment.
The present invention also provides a method for treating immune diseases, which comprises administering one or more proteins described above in an effective amount to a patient in need of such treatment.
The present invention also provides a method for stimulating IgE production, which comprises administering one or more proteins described above in an effective amount to a patient in need of such treatment.
The present invention also provides a method for treating allergic diseases, which comprises administering one or more proteins described above in an effective amount to a patient in need of such treatment.
The present invention also relates to the use of one or more proteins described above in the production of immunomodulating agents.
The present invention also relates to the use of one or more proteins described above in the production of therapeutic agents for immune diseases.
The present invention also relates to the use of one or more proteins described above in the production of IgE production-stimulating agents.
The present invention also relates to the use of one or more proteins described above in the production of therapeutic agents for allergic diseases.
The present invention will be further described below.
As used herein, xe2x80x9cimmunomodulatory activityxe2x80x9d means stimulation of non-specific immunoglobulin production from B cells and modulation of immune responses mediated by Th1 and Th2.
As used herein, xe2x80x9cstimulation of non-specific immunoglobulin production from B cellsxe2x80x9d means that B cells are stimulated to produce non-specific immunoglobulins (Ig), particularly non-specific IgE, not to produce immunoglobulins against specific antigens. When producing Ig, in general, B cells should be converted into blast cells (i.e., blast formation) upon stimulation by antigen-presenting cells which present specific antigens on their surface. However, the proteins of the present invention do not cause the blast formation, so that mature B cells proliferate and thus produce non-specific Ig.
As used herein, xe2x80x9cmodulation of immune responses mediated by Th1 and Th2xe2x80x9d means that the immune response pattern is changed from cellular immunity into humoral immunity and vice versa by inhibiting cytokine production from each T cell subset Th1 and Th2 or by inducing cytokines from one subset that inhibit cytokines produced from the other subset.
As used herein, xe2x80x9cimmune diseasexe2x80x9d refers to a disease resulting from dysfunction of the immune system, one of defense mechanisms in the body, including diseases caused by both abnormal humoral and cellular immunity. This term also includes autoimmune diseases caused by autoantibody, autosensitized lymphocyte or immune complex, as well as graft versus host disease caused by graft versus host reaction (GVH reaction) in which graft rejection occurs. Allergic diseases and the like are also included.
As used herein, xe2x80x9cTh1-dominant autoimmune diseasexe2x80x9d refers to an autoimmune disease showing increased cytokine production from Th1 cells, including IFN-xcex3, IL-2, GM-CSF, TNF-xcex1, and IL-3. Specific examples include multiple sclerosis, insulin-dependent diabetes mellitus, Crohn""s disease, uveitis, chronic rheumatism, and systemic lupus erythematosus.
As used herein, xe2x80x9cautoimmune disease not known to be Th1-dominantxe2x80x9d refers to an autoimmune disease that is not known to show increased cytokine production from Th1 cells. Specific examples include scleroderma, multiple myositis, vasculitis syndrome, mixed connective tissue disease, Sjxc3x6gren""s syndrome, hyperthyroidism, Hashimoto""s disease, myasthenia gravis, Guillain-Barrxc3xa9syndrome, autoimmune hepatopathy, ulcerative colitis, autoimmune nephropathy, autoimmune hematopathy, idiopathic interstitial pneumonia, hypersensitivity pneumonitis, autoimmune dermatosis, autoimmune cardiopathy, autoimmune infertility, and Behcet""s disease.
As used herein, xe2x80x9callergic diseasexe2x80x9d refers to a disease associated with allergic reaction. Specific examples include chronic bronchitis, atopic dermatitis, pollinosis (allergic rhinitis), allergic angiitis, allergic conjunctivitis, allergic gastroenteritis, allergic hepatopathy, allergic cystitis, and allergic purpura.
As used herein, xe2x80x9cdeletion, substitution or addition of one or more amino acidsxe2x80x9d or xe2x80x9cone or more amino acids are deleted, substituted or addedxe2x80x9d means both naturally occurring modification and artificially introduced modification using site-directed mutagenesis (Nucleic Acids Research, Vol. 10, No. 20, pp. 6487-6500, 1982) etc.
The immunomodulatory proteins of the present invention have the above formula (I): X-Y-Z. In the formula, X represents an amino acid sequence of SEQ ID NO: 1 or 2, each of Y and Z is absent or represents an amino acid sequence of SEQ ID NO: 1 or 2. The amino acid sequence of SEQ ID NO: 1 is hereinafter designated V1, while the amino acid sequence of SEQ ID NO: 2 is designated V2.
When expressed using V1 and V2, the proteins of the present invention encompass V1 (SEQ ID NO: 1), V2 (SEQ ID NO: 2), V1+V2 (SEQ ID NO: 3), V2+V1 (SEQ ID NO: 4), V1+V2+V1 (SEQ ID NO: 5), V2+V1+V2 (SEQ ID NO: 6), V1+V1 (SEQ ID NO: 7), V2+V2 (SEQ ID NO: 8), V1+V1+V1 (SEQ ID NO: 9), V1+V1+V2 (SEQ ID NO: 10), V1+V2+V2 (SEQ ID NO: 11), V2+V2+V2 (SEQ ID NO: 12), V2+V2+V1 (SEQ ID NO: 13), and V2+V1+V1 (SEQ ID NO: 14).
The amino acid sequence homology between V1 and V2 is relatively high, and amino acids 1-61 in these sequences are homologous to each other. This homologous sequence is shown in SEQ ID NO: 15.
Each of V1 and V2 of the present invention is protein originated from Dirofilaria immitis, a kind of parasitic helminth. The protein from Dirofilaria immitis may be obtained by combining the following techniques: extraction of proteins from Dirofilaria immitis, anion exchange chromatography, and gel filtration chromatography etc.
The protein from Dirofilaria immitis, hereinafter designated DiNCF, has neutrophil chemotactic activity and comprises a repeated sequence in which two types of tens of molecules DiNCF V1 and DiNCF V2 are repeated in tandem. Ohashi et al. have cloned cDNA for DiNCF (Ohashi, supra) and thus amino acid sequences for DiNCF V1 and DiNCF V2 have been determined.
These amino acid sequences correspond to V1 and V2, respectively. V1 contains 129 amino acids, while V2 contains 131 amino acids (SEQ ID NO: 1 and 2).
The proteins of the present invention may encompass heterodimer and heterotrimer, in which V1 and V2 are repeated in tandem, as well as homodimer and homotrimer, in which one of V1 or V2 is repeated in tandem. The homodimer and homotrimer are both non-naturally occurring proteins.
In these two proteins, their N-terminal sides, i.e., amino acids 1 to 61 are homologous to each other, but the rest of amino acids are less homologous to each other.
The proteins of the present invention may encompass not only proteins having full-length sequences of V1 and V2, but also a protein having the amino acid sequence of SEQ ID NO: 15.
The amino acid sequence of SEQ ID NO: 15 corresponds to amino acids 1-61 of V1 and V2, and may be modified by substitution, deletion or addition of one or more amino acids so long as the resulting protein has immunomodulatory activity.
Further, preferred amino acid sequences comprise amino acids 1-76 of the amino acid sequence depicted in SEQ ID NO: 1 or 2.
These amino acid sequences may be modified by substitution, deletion or addition of one or more amino acids in the same manner for the protein having the amino acid sequence of SEQ ID NO: 15.
To obtain a gene encoding the protein of the present invention, a vector carrying DiNCF gene may be subjected to PCR method using primers, each of which includes an appropriate restriction site, to amplify the gene of interest; the vector may be treated with an appropriate restriction enzyme to isolate a DNA fragment having the desired molecular weight, which may be then ligated to an appropriate linker; or each DNA encoding DiNCF V1 or DiNCF V2 may be chemically synthesized based on the nucleotide sequence of the gene encoding Dirofilaria immitis protein according to standard techniques and these DNAs may then be optionally joined to one another to form the sequences listed above.
The DNA fragment thus obtained, i.e., the gene encoding the protein of the present invention, may be ligated to an appropriate vector treated with a restriction enzyme to obtain a recombinant vector for protein expression.
This recombinant vector may be used to transform an appropriate host. The desired transformant may be selected and then grown to obtain a vector producing the protein of interest.
Examples of the vector carrying DiNCF gene include vectors pDi6, pDi18 and pD-4 that carry the nucleotide sequences of DNAs encoding DiNCF V1 and DiNCF V2, each of which has been constructed simultaneously with clone pD-4 constructed for the purpose of cloning DiNCF (see Maruyama, H. et al., 30 (14): 1315-1320, 1993). The vector pDi6 may be preferably used because it contains both V1 and V2 genes.
The vector pDi6 was constructed according to the method described in Ohashi (Ohashi M. et al., Mol. Immunol. 30 (14):1315-1320, 1993). Namely, adult Dirofilaria immitis is collected from the heart of a dog naturally infected with Dirofilaria immitis and cut into small pieces on ice, from which mRNA is then purified using mRNA Purification Kit (Pharmacia).
cDNA is synthesized from this mRNA using cDNA Synthesis Kit (Pharmacia) and then inserted into an EcoR I site of xcexgt11 vector (Promega Biotec., Madison, Wis., USA). DNA of this cDNA-inserted xcexgt11 is subjected to in vitro packaging using Gigapack (Stratagene, La Jolla, Calif., USA) to construct a cDNA library.
The clone of interest is selected from this cDNA library using an anti-DiNCF antibody according to the method described in Huynh, T. V. et al., A Practical Approach, Vol. 1, pp. 49-78, Glover D. M. ed., IRL Press, Oxford.
The selected clone is digested with EcoR I to excise the inserted gene from the phage DNA. The excised gene is then inserted into EcoR I site of phagemid vector pBluescript SK (xe2x88x92) to construct the plasmid pDi6.
To obtain a DNA, e.g., V1+V2, in which these genes are repeated in tandem, restriction enzyme NspV may be preferably used because the consensus sequence has only one site for this enzyme to recognize.
Alternatively, the chemical synthesis of the above DNA fragment may be carried out in a known manner such as phosphoramidite method or phosphit-triester method.
The vector used for construction of the recombinant vector for protein expression includes plasmid vectors such as pET3a, pTrc and pKK2B-3; phage vectors such as xcexgt11 and M13; pBluescript II; or phagemid vectors such as pcDNA2.1. The plasmid vector pET3a may be preferably used because it leads to good yield.
The host to be transformed includes E. coli and the like. E. coli may be preferably used because of its simple manipulation, particularly HMS 174 (DE3) strain may provide a high protein expression efficiency.
The host may be transformed using standard techniques including, but not limited to, calcium chloride method or electroporation method, both of which are preferred due to their high transformation efficiency.
The transformants obtained by transformation with the vector carrying the gene encoding the protein of the present invention may be screened for their antibiotic resistance. The antibiotic used as a screening marker includes ampicillin, tetracycline, kanamycin and the like. Ampicillin may be preferably used because the preferred vector is pET3a.
The selected transformant may be grown to obtain the protein of the present invention.
The DNA fragment having the nucleotide sequence encoding the protein of the present invention may be modified by site-directed mutagenesis to introduce a mutation at any position in the DNA fragment. Such a DNA modification can provide various modified proteins.
The present invention will be further described in terms of sequence V1.
Plasmid vector pDi6 carrying a gene encoding DiNCF V1 region (distributed from Prof. Makoto Ohashi of Tokushima University) is used as a template. PCR amplification is carried out using this template vector and primers, each including a restriction site and a stop codon. By using such a primer including a restriction site, the fragment of interest can be introduced into an expression vector downstream from its initiation codon in the desired orientation and in frame. This enables the protein of interest to be expressed.
The use of the following primers achieve an efficient amplification of the nucleotide sequence of interest:
N-terminal primer:
5xe2x80x2-GCATATGAATGATCATAATTTAGAAAGC-3xe2x80x2 (SEQ ID NO: 16), and
C-terminal primer:
5xe2x80x2-CTAAAGGATCCTATCACCGCTTACGCCGTTCATTCATTG-3xe2x80x2 (SEQ ID NO: 17).
These primers may be chemically synthesized, for example, by phosphoramidite method, or we may ask a company (e.g. Biologica Co.) to synthesize these primers.
PCR may be carried out using the above primers, DiNCF V1 as a template, Ex Taq DNA polymerase, and a buffer and dNTP (equivalent mixture of dATP, dGTP, dCTP, dTTP) contained in Ex Taq Kit (Takara Shuzo Co., Ltd.) etc.
The resulting amplified fragment may be purified by MicroSpin Column and the like, digested with Nde I and BamH I, purified again, and then inserted into an expression vector. For this purpose, pET3a is digested with Nde I and BamH I and purified using MicroSpin Column S400 (Pharmacia) to obtain its main segment as an expression vector.
The above PCR amplified fragment is ligated to this digested pET3a using DNA ligation kit (Takara Shuzo Co., Ltd.) according to the manufacture""s instructions to obtain the circular DNA of interest.
The circular DNA is introduced into E. coli strain JM109 by calcium chloride method to transform the strain JM109. The resulting transformants are cultured in LB medium with ampicillin. Cells grown in the medium are collected by centrifugation and subjected to alkaline SDS method to isolate and obtain a plasmid, designated pDP5.
The nucleotide sequence of the resulting plasmid may be examined using Sequenase kit (United States Biochemical Corporation, USA) in order to confirm whether the insert of interest is inserted correctly in the vector and whether any unexpected changes are observed before and after the insert.
To obtain the dimer or trimer protein of the present invention, an expression vector may be constructed as described below in terms of dimer V1+V1.
The plasmid pDP5 constructed as described above is digested with Nsp V, treated with phenol by standard techniques, dephosphorylated using calf intestine alkaline phosphatase (CIP), and then treated with phenol in order to deactivate the enzyme.
Meanwhile, the plasmid pDi6 is digested with the same restriction enzyme and electrophoresed on agarose gel to purify a band of the desired molecular weight. GENECLEAN II kit (Funakoshi Co., Ltd.) etc. may be used for this purpose.
This purified fragment is ligated to the linearized vector treated with CIP in the same manner as described above. The ligated DNA is used to transform E. coli strain JM109 to obtain transformants.
The transformed clones are picked up properly and cultured overnight in a medium with ampicillin. Cells grown in the medium are collected by centrifugation, from which vector DNAs are then isolated and purified by alkaline SDS method. Each of the vectors thus obtained is digested with an appropriate restriction enzyme and analyzed by electrophoresis to obtain the vector of interest.
This vector is used to transform E. coli HMS 174 (DE3) in the same manner as described above. Each of the resulting transformants is cultured until the absorbance A550 becomes 0.8. When the absorbance A550 becomes 0.8, the culture further continues with addition of IPTG (isopropylthiogalactoside).
The cells are separated from the culture fluid by centrifugation, suspended in a solution containing 8M urea and 0.1M Tris-HCl (pH 7.0), and then ultrasonically treated and centrifuged to obtain the supernatant.
This supernatant is subjected to SDS polyacrylamide electrophoresis using Phast System (Pharmacia). A control is E. coli strain transformed with normal pET3a.
The production of the desired protein can be confirmed by the above procedures.
Each of the transformants thus obtained is cultured and grown cells are collected by centrifugation. A predetermined amount of the cells is extracted with hydrochloric acid to obtain a hydrochloric acid extract.
After neutralization with sodium hydroxide, this extract is mixed with ammonium sulfate and centrifuged to separate precipitated products. These precipitated products are dissolved in PBS (physiological phosphate buffer) and purified by gel filtration chromatography to obtain the final purified product.
The proteins thus obtained can also be used to prepare DNAs having nucleotide sequences encoding these proteins according to various known techniques. In the present invention, DNAs having these nucleotide sequences may be modified to introduce substitution, deletion, addition or insertion of one or more bases, for example, according to site-directed mutagenesis (Zoller et al., Nucleic Acids Research, Vol. 10, No. 20, pp. 6487-6500, 1982). Those skilled in the art may easily prepare these modified DNAs. The present invention can encompass these DNAs so long as proteins encoded by them have immunomodulatory activity.
Physiological functions of these proteins may be examined as follows. A mouse is sacrified by dislocating its cervical vertebra and its spleen is excised to obtain lymphocytes. These splenic lymphocytes are centrifuged to remove the supernatant, washed with ACT solution (0.83% NH4Cl, 170 mM Tris-HCl (pH 7.6)), and then suspended in RPMI 1640 medium with fetal calf serum (FCS) to form splenic lymphocyte suspension.
Next, B cells are prepared as follows. These splenic lymphocytes are incubated with an anti-Thy-1.2 antibody added thereto at 4xc2x0 C., and then suspended in RPMI 1640 medium with 5% fetal calf serum (FCS) after washing. This suspension is mixed well and reacted with a commercially available complement solution prepared from rabbit etc. at about 37xc2x0 C. for about one hour. After washing, these lymphocytes are suspended again in RPMI 1640 medium with 5% FCS at a predetermined cell density (B cell suspension). The cell density suitable for proliferative response to stimulation is 1xc3x97105 cells/mL to 5xc3x97106 cells/mL.
The proliferative response to stimulation may be confirmed as follows.
The B cell suspension prepared as described above is divided into each well of a 24-well plate and cultured in RPMI 1640 medium with 5% FCS for a predetermined period. Each protein of the present invention is added at a predetermined concentration and the culture continues for additional 48 hours in order to perform MTT assay. The protein of the preset invention is used preferably at a concentration of 0.1 to 1,000 xcexcg/nL because this concentration range provides a significant response in MTT assay, more preferably 10 to 100 xcexcg/mL because a higher response can be observed.
MTT assay may be performed as follows. The cell suspension (500 xcexcl) is incubated and reacted with MTT solution (50 xcexcl) added thereto at 37xc2x0 C. for 4 hours, and then incubated with stop solution (450 xcexcl) added thereto at room temperature for 30 minutes in order to stop the reaction. Absorbances at 630 nm and 570 nm (designated A630 and A570, respectively) are measured to determine the grade of proliferative response.
The MTT solution is prepared by dissolving MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenylterazolium bromide, Sigma) in PBS at a concentration of 5 mg/mL. The stop solution is isopropanol containing 0.04 N hydrochloric acid.
For a blank experiment, the medium alone is subjected to the same treatment as described above and tested for its absorbances at 630 nm and 570 nm (designated A0630 and A0570, respectively). MTT assay value can be calculated based on these measured values using the following equation:
MTT assay value=(A570xe2x88x92A630)xe2x88x92(A0570=A0630).
Lipopolysaccharide (LPS) may be used as a positive control.
Each of the peptides according to the present invention may be tested for its ability to induce IgE production as follows. For example, a mouse is administered intraperitoneally with a mixture of DiNCF V1 in a predetermined amount and aluminum hydroxide adjuvant (ALUM).
Blood is taken from its caudal artery with a heparinized tube before administration and on the 7th, 14th and 21st days after administration. Plasma is separated and IgE contained therein is detected by enzyme antibody technique. Enzyme antibody technique may be carried out according to, but not limited to, the following procedures.
The following materials are used in this technique: anti-DNP IgE as a standard IgE, anti-mouse IgE Fc xcex5 rat monoclonal antibody as a primary antibody, peroxidase-labeled anti-mouse IgE polyclonal antibody as a secondary antibody, an appropriate blocking agent, reaction buffer such as PBS containing 0.1% bovine serum albumin, and PBS-Tween containing 0.05% Tween 20 in PBS.
The primary antibody is diluted with sodium carbonate buffer (pH 9.5) to a predetermined concentration and bound to the surface of each well in a 96-well microtiter plate. Each well is blocked using blocking solution and then washed with PBS-Tween.
The protein of the present invention or the standard IgE is added to each well, for example, in an amount of 100 xcexcL/well, and incubated at room temperature for an appropriate period, e.g., 3 hours. The plate is washed with PBS-Tween 2 or 3 times. The secondary antibody is optionally diluted with the reaction buffer and added to each well in an amount of 100 xcexcL/well. Incubation continues at room temperature for about 3 hours. The plate is washed with PBS-Tween.
Substrate solution is then added to each well and incubated in the dark for a few minutes to develop color. Upon development of a detectable color, stop solution is added to each well. The plate is then read at 490 nm using a microplate reader to calculate the concentration of IgE contained in plasma based on calibration curve prepared from the standard IgE.
Each peptide may be tested for its in vivo immunomodulatory activity using a rat model with autoimmune encephalomyelitis.
Namely, the protein of the present invention is administered to a rat via the footpads of its hind legs in an amount of 10 to 1,000 xcexcg/rat, preferably 100 xcexcg/rat. Control group is similarly administered with PBS. This administration continues for 41 days. On the 41st day after the administration has started, both test and control groups are further administered with guinea pig myelin basic protein peptide (GPE) emulsified with an appropriate adjuvant in an amount of about 2 to 10 xcexcg/rat, preferably 5 xcexcg/rat. Preferred adjuvants include killed Mycobacterium tuberculosis and the like.
On the 14th day after the GPE administration, changes in clinical signs of both groups are scored in accordance with the following criteria shown in Table 1.
Each protein of the present invention tested for its immunomodulatory activity may be formulated with each pharmaceutically acceptable ingredient to form a therapeutic agent for immune diseases. The pharmaceutically acceptable ingredient includes excipient, binder, disintegrating agent, coloring agent, flavoring agent, corrective, solubilizing agent, emulsifying agent, preservative, suspending agent, stabilizing agent, isotonizing agent, and buffer.
Specifically, the excipient includes starch or lactose for solid formulation, and water for liquid formulation. The binder includes gum Arabic, starch, carboxymethylcellulose sodium (CMC-Na), water, ethanol, and simple syrup. The disintegrating agent includes various surfactants, carbonate or the like. The coloring agent includes natural ones and synthetic ones acceptable under Food Sanitation Law.
The flavoring agent includes various essential oils such as orange oil, lemon oil and coriander oil. The corrective includes simple syrup and saccharose. The solubilizing agent includes polyoxyethylene hydrogenated castor oil derivatives, sodium benzoate and ethylenediamine.
The emulsifying agent includes various types of Span such as Span 20 and Span 60, various types of Tween such as Tween 20 and Tween 80. The preservative includes phenol, thimerosal and chlorobutanol.
The suspending agent includes CMC-Na, methylcellulose, simple syrup and glycerine. The stabilizing agent includes albumin, gelatin and sorbitol.
The isotonizing agent includes glucose and sodium chloride. The buffer includes phosphates.
These ingredients may be used alone or in combination for formulation.
The peptide of the present invention may be formulated in any dosage form including, but not limited to, tablet, granule, capsule, injection or the like.
Since the protein of the present invention affects T cell subset, Th2, as described above, immune diseases that can be treated using the therapeutic agent for immune diseases may be not only those thought to be Th1-dominant, but also those not known to be Th1-dominant. Specifically, the immune diseases thought to be Th1-dominant include multiple sclerosis, insulin-dependent diabetes mellitus, Crohn""s disease, uveitis, chronic rheumatism, and systemic lupus erythematosus.
The immune diseases not known to be Th1-dominant include scleroderma, multiple myositis, vasculitis syndrome, mixed connective tissue disease, Sjxc3x6gren""s syndrome, hyperthyroidism, Hashimoto""s disease, myasthenia gravis, Guillain-Barrxc3xa9syndrome, autoimmune hepatopathy, ulcerative colitis, autoimmune nephropathy, autoimmune hematopathy, idiopathic interstitial pneumonia, hypersensitivity pneumonitis, autoimmune dermatosis, autoimmune cardiopathy, autoimmune infertility, and Behcet""s disease.
The protein of the present invention may also be used as an IgE production-stimulating agent for treatment of allergic diseases. That is, it enables mature B cells, i.e., polyclonal B cells to proliferate, thereby inducing increased non-specific IgE production, but not inducing monoclonal IgE production through blast formation as usually observed during elevation of IgE level.
When the protein of the present invention is used as an IgE production-stimulating agent, allergic diseases to be treated include chronic bronchitis, atopic dermatitis, pollinosis (allergic rhinitis), allergic angiitis, allergic conjunctivitis, allergic gastroenteritis, allergic hepatopathy, allergic cystitis, and allergic purpura.
The IgE production-stimulating agent comprising the protein of the present invention may also be used for treatment of rejection reaction occurring in organ transplantation. As used herein, xe2x80x9corgan transplantationxe2x80x9d refers to transplantation of organs including kidney, liver, lung and heart. Other organs such as bone, skin and tendon may also be included. The IgE production-stimulating agent of the present invention can alleviate rejection reaction occurring after the organ transplantation because it stimulates the production of non-specific IgE.
A formulation example is shown below using V1+V1 among the proteins of the present invention.
(Injection Formulation)
10 mM phosphate buffer (9 mL, pH 7.4) as a buffer and human serum albumin (10 mg) as a stabilizing agent were added to and dissolved in 1 mg/mL V1+V1 solution. The resulting solution was dispensed 1 mL into 5 mL glass vials.
V1+V1 0.1 mg
Phosphate buffer 0.9 mL
Human serum albumin 1 mg
Each vial was lyophilized at xe2x88x9220xc2x0 C. and then sealed to obtain injection formulation. When used, this injection formulation may be reconstituted with 1 mL of distilled water for injection (Otsuka Pharmaceutical Co., Ltd.).
This specification includes part or all of the contents as disclosed in the specification and/or drawings of Japanese Patent Application No. 10-87189, which is a priority document of the present application.